intro to ms yates - university of pittsburgh · 365.23 22.27 444.74 20.58 690.33 21.06 436.76 21.80...
TRANSCRIPT
Introduc)on to Mass Spectrometry
Nathan A. Yates University of Pi8sburgh
Biomedical Mass Spectrometry Center
Structural Biophysics Mass Spec Lecture I Pi8sburgh PA, December 3, 2014
1
Overview
• Informa1on contained in a mass spectrum • Components of a mass spectrometer • MS analysis of biomolecules • Complex mixture analysis and data processing • Trace level analysis by tandem mass spectrometry (MS/MS) • Shotgun Proteomics • Global analysis of complex protein mixtures • Orbitrap mass analyzers
• Differen1al mass spectrometry
Focus: Introduction to Mass Spectrometry and Methods for the Structural an Quantitative Analysis of Biomolecules.
2
Mass Spectrometry
3
A Mass Spectrum
m/z =18 (H20)+ since O + H + H = 16 + 1 + 1 = 18 amu m/z=17 (HO)+ since O + H = 16 + 1 = 17 amu m/z=16 (O)+ since O = 16 amu m/z=1 (H)+ since H = 1 amu
4
Electron Impact Method of Ioniza)on
M + e-‐ M+. 2e-‐ +
F+ N + M+.
5
Components of a Mass Spectrometer
Ion Source Mass Analyzer Detector
Data System
6
MS Analysis of Biomolecules?
Simvasta)n 418.6 Da
Myoglobin 16,955 Da
7
Simvasta)n Mass Spectrum (Electron Impact Ioniza)on)
8
9
Proteomic Technology -‐ Electrospray Ioniza)on Produces Gas Phase Ions from Solu)on Phase Analytes (e.g. Pep)des and Proteins)
Electrospray Ioniza)on
John B. Fenn -‐ 2002 Nobel Prize in Chemistry for the development of so` desorp)on ionisa)on methods for mass spectrometric analyses of biological macromolecules
To MS
From µLC (M+H)+
Protonated Molecules
Simvasta)n ESI Mass Spectrum (Electrospray Ioniza)on)
MW = 418.6 Da C25H38O5
ESI-‐
ESI+
10
High and Low Resolu)on MS
C25h38o5 +Na: C25 H38 O5 Na1 p(gss, s/p:40) Chrg 1...
440 445 450m/z
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441.26
442.27
443.27445.28
C25h38o5 +Na: C25 H38 O5 Na1 p(gss, s/p:40) Chrg 1...
442 444 446 448m/z
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441.26
442.26
443.27444.27 445.27
Unit Resolu)on 1 Da @ 10% FWHM
High Resolu)on 0.01 Da @ 10% FWHM
11
Myoglobin Mass Spectrum (Electrospray Ioniza)on)
Myoglobin 16,955 Da
12
Mixture Analysis by (LC-‐MS)
13
What about complex biological mixtures?
14
LC-‐MS Data Analysis C:\Users\yatesn\Desktop\q1652 7/23/2012 11:04:10 AM
RT: 0.00 - 85.01 SM: 3B
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38.60789.43
28.89687.86
29.10687.86 42.77
910.45 63.56687.3228.54
687.86 74.431009.0024.56
844.4431.97442.26
42.92693.87
61.19790.39
64.59724.40
19.53609.31
14.32423.22 70.15
1104.5545.77678.31
58.00876.89 74.64
1455.2020.00466.73 55.79
725.8613.36339.18 53.88
515.79 75.111086.88
7.53402.712.69
312.6980.84445.12
NL:3.05E9Base Peak F: FTMS + p NSI Full ms [300.00-1500.00] MS q1652
q1652 #7077-7835 RT: 19.20-20.72 AV: 91 NL: 2.20E8T: FTMS + p NSI Full ms [300.00-1500.00]
300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z
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609.31466.73
435.27 522.29932.45392.73 819.37
690.33
784.45534.34369.20 847.48671.42 713.42575.30 1217.61645.35 914.44 1047.50946.49
1132.58 1379.661251.66 1462.59
15
Narrow in on a por)on of the mass spectrum (m/z = 400-‐450)
C:\Users\yatesn\Desktop\q1652 7/23/2012 11:04:10 AM
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38.60789.43
28.89687.86
29.10687.86 42.77
910.45 63.56687.3228.54
687.86 74.431009.0024.56
844.4431.97442.26
42.92693.87
61.19790.39
64.59724.40
19.53609.31
14.32423.22 70.15
1104.5545.77678.31
58.00876.89 74.64
1455.2020.00466.73 55.79
725.8613.36339.18 53.88
515.79 75.111086.88
7.53402.712.69
312.6980.84445.12
NL:3.05E9Base Peak F: FTMS + p NSI Full ms [300.00-1500.00] MS q1652
q1652 #7077-7835 RT: 19.20-20.72 AV: 91 NL: 1.11E8T: FTMS + p NSI Full ms [300.00-1500.00]
400 405 410 415 420 425 430 435 440 445m/z
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435.27
430.28424.25414.75 436.27
440.25424.75423.73407.26 444.74415.25 442.22402.27 431.28428.58 445.24400.56 417.89 437.73425.25408.23
433.22
421.21410.21403.27414.22
16
Plot a Selected Ion Chromatogram (418+) C:\Users\yatesn\Desktop\q1652 7/23/2012 11:04:10 AM
RT: 0.00 - 85.01 SM: 3B
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38.60789.4328.89
687.86 31.81442.26
42.77910.45
63.56687.32
28.54687.86
74.431009.00
24.56844.44
61.19790.39
44.54644.86
19.53609.31
14.32423.22
64.69724.4155.79
725.8611.53389.19
75.111086.88
8.14395.23
34.09417.73 35.12
417.7339.74417.7412.47
418.2218.25417.56
20.46417.88 28.13
418.2442.46417.72
50.96418.25
11.99418.20
NL: 3.05E9Base Peak F: FTMS + p NSI Full ms [300.00-1500.00] MS q1652
NL: 8.47E8m/z= 417.50-418.50 F: FTMS + p NSI Full ms [300.00-1500.00] MS q1652
q1652 #7077-7835 RT: 19.20-20.72 AV: 91 NL: 1.11E8T: FTMS + p NSI Full ms [300.00-1500.00]
400 405 410 415 420 425 430 435 440 445m/z
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435.27
430.28424.25414.75 436.27
440.25424.75423.73407.26 444.74415.25 442.22402.27 431.28428.58 445.24400.56 417.89 437.73425.25408.23
433.22
421.21410.21403.27414.22
17
Narrow the chromatographic )me range (19-‐22min) C:\Users\yatesn\Desktop\q1652 7/23/2012 11:04:10 AM
RT: 18.94 - 22.68 SM: 3B
19.0 19.5 20.0 20.5 21.0 21.5 22.0 22.5Time (min)
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19.53609.31
20.00466.73 20.09
466.7319.40392.73
19.66609.31
21.42419.72
21.37419.72 22.39
815.4319.13365.23
22.27444.74
20.58690.33
21.06436.76 21.80
488.73
20.46417.8820.00
417.89 20.85417.88 21.21
417.55
20.11417.89
21.82417.88
19.66418.24
22.14417.72
19.17418.26
22.42417.87
19.31417.74
NL: 1.35E9Base Peak F: FTMS + p NSI Full ms [300.00-1500.00] MS q1652
NL: 1.28E8m/z= 417.50-418.50 F: FTMS + p NSI Full ms [300.00-1500.00] MS q1652
q1652 #7077-7835 RT: 19.20-20.72 AV: 91 NL: 1.11E8T: FTMS + p NSI Full ms [300.00-1500.00]
400 405 410 415 420 425 430 435 440 445m/z
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435.27
430.28424.25414.75 436.27
440.25424.75423.73407.26 444.74415.25 442.22402.27 431.28428.58 445.24400.56 417.89 437.73425.25408.23
433.22
421.21410.21403.27414.22
18
Plot an average spectrum for a chromatographic peak of interest (RT=20.0min)
C:\Users\yatesn\Desktop\q1652 7/23/2012 11:04:10 AM
RT: 18.94 - 22.68 SM: 3B
19.0 19.5 20.0 20.5 21.0 21.5 22.0 22.5Time (min)
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19.53609.31
20.00466.73 20.09
466.7319.40392.73
19.66609.31
21.42419.72
21.37419.72 22.39
815.4319.13365.23
22.27444.74
20.58690.33
21.06436.76 21.80
488.73
20.46417.8820.00
417.89 20.85417.88 21.21
417.55
20.11417.89
21.82417.88
19.66418.24
22.14417.72
19.17418.26
22.42417.87
19.31417.74
NL: 1.35E9Base Peak F: FTMS + p NSI Full ms [300.00-1500.00] MS q1652
NL: 1.28E8m/z= 417.50-418.50 F: FTMS + p NSI Full ms [300.00-1500.00] MS q1652
q1652 #7465-7497 RT: 20.00-20.02 AV: 2 NL: 7.19E7T: FTMS + p NSI Full ms [300.00-1500.00]
400 405 410 415 420 425 430 435 440 445m/z
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428.58 430.22 435.27417.89
408.52
418.23
407.26430.72
408.85435.78
433.22445.73417.24 418.56 436.28421.25409.73 425.20416.21 423.74402.27400.23 411.75 418.90 444.29404.73 436.78 442.26426.20415.24397.25
406.74
19
Narrow in on a por)on of the mass spectrum (m/z = 415-‐420)
C:\Users\yatesn\Desktop\q1652 7/23/2012 11:04:10 AM
RT: 18.94 - 22.68 SM: 3B
19.0 19.5 20.0 20.5 21.0 21.5 22.0 22.5Time (min)
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19.53609.31
20.00466.73 20.09
466.7319.40392.73
19.66609.31
21.42419.72
21.37419.72 22.39
815.4319.13365.23
22.27444.74
20.58690.33
21.06436.76 21.80
488.73
20.46417.8820.00
417.89 20.85417.88 21.21
417.55
20.11417.89
21.82417.88
19.66418.24
22.14417.72
19.17418.26
22.42417.87
19.31417.74
NL: 1.35E9Base Peak F: FTMS + p NSI Full ms [300.00-1500.00] MS q1652
NL: 1.28E8m/z= 417.50-418.50 F: FTMS + p NSI Full ms [300.00-1500.00] MS q1652
q1652 #7465-7497 RT: 20.00-20.02 AV: 2 NL: 6.42E7T: FTMS + p NSI Full ms [300.00-1500.00]
415.0 415.5 416.0 416.5 417.0 417.5 418.0 418.5 419.0 419.5 420.0m/z
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417.89
418.23
417.24 418.56416.21
418.90416.71416.26 419.23415.24 416.89415.74 417.81 419.70
20
Plot an average spectrum for a chromatographic peak of interest (RT=20.8min)
C:\Users\yatesn\Desktop\q1652 7/23/2012 11:04:10 AM
RT: 18.94 - 22.68 SM: 3B
19.0 19.5 20.0 20.5 21.0 21.5 22.0 22.5Time (min)
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19.53609.31
20.00466.73 20.09
466.7319.40392.73
19.66609.31
21.42419.72
21.37419.72 22.39
815.4319.13365.23
22.27444.74
20.58690.33
21.06436.76 21.80
488.73
20.46417.88
20.00417.89 20.85
417.8821.21417.8920.11
417.8921.84417.88
22.12417.88
NL: 1.35E9Base Peak F: FTMS + p NSI Full ms [300.00-1500.00] MS q1652
NL: 7.94E7m/z= 417.87-417.91 F: FTMS + p NSI Full ms [300.00-1500.00] MS q1652
q1652 #7896 RT: 20.85 AV: 1 NL: 4.31E7T: FTMS + p NSI Full ms [300.00-1500.00]
415.0 415.5 416.0 416.5 417.0 417.5 418.0 418.5 419.0 419.5 420.0m/z
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417.88
418.22
418.55
416.23 418.89417.23419.20417.73415.23 416.73 417.26415.74 416.96414.91 418.26
21
Narrow in on a por)on of the mass spectrum (m/z = 418.1-‐418.3)
C:\Users\yatesn\Desktop\q1652 7/23/2012 11:04:10 AM
RT: 18.94 - 22.68 SM: 3B
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19.53609.31
20.00466.73 20.09
466.7319.40392.73
19.66609.31
21.42419.72
21.37419.72 22.39
815.4319.13365.23
22.27444.74
20.58690.33
21.06436.76 21.80
488.73
20.46417.88
20.00417.89 20.85
417.8821.21417.8920.11
417.8921.84417.88
22.12417.88
NL: 1.35E9Base Peak F: FTMS + p NSI Full ms [300.00-1500.00] MS q1652
NL: 7.94E7m/z= 417.87-417.91 F: FTMS + p NSI Full ms [300.00-1500.00] MS q1652
q1652 #7896 RT: 20.85 AV: 1 NL: 2.49E7T: FTMS + p NSI Full ms [300.00-1500.00]
418.10 418.12 418.14 418.16 418.18 418.20 418.22 418.24 418.26 418.28 418.30 418.32 418.34m/z
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418.20
418.23 418.2622
Plot a selected ion chromatogram for m/z 418.20 C:\Users\yatesn\Desktop\q1652 7/23/2012 11:04:10 AM
RT: 18.94 - 22.68 SM: 3B
19.0 19.5 20.0 20.5 21.0 21.5 22.0 22.5Time (min)
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19.53609.31
20.00466.73 20.09
466.7319.40392.73
19.66609.31
21.42419.72
21.37419.72 22.39
815.4319.13365.23
22.27444.74
20.58690.33
21.06436.76 21.80
488.73
20.83418.20
20.92418.20
19.66418.20 22.40
418.2019.73418.20
NL: 1.35E9Base Peak F: FTMS + p NSI Full ms [300.00-1500.00] MS q1652
NL: 2.11E7m/z= 418.20-418.21 F: FTMS + p NSI Full ms [300.00-1500.00] MS q1652
q1652 #7896 RT: 20.85 AV: 1 NL: 2.49E7T: FTMS + p NSI Full ms [300.00-1500.00]
418.10 418.12 418.14 418.16 418.18 418.20 418.22 418.24 418.26 418.28 418.30 418.32 418.34m/z
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418.20
418.23 418.26
23
Expand view to see full )me range C:\Users\yatesn\Desktop\q1652 7/23/2012 11:04:10 AM
RT: 0.00 - 85.01 SM: 3B
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38.60789.4328.89
687.86 31.81442.26
42.77910.45
63.56687.32
28.54687.86
74.431009.00
24.56844.44
61.19790.39
44.54644.86
19.53609.31
14.32423.22
64.69724.4155.79
725.8611.53389.19
75.111086.88
8.14395.23
20.83418.20
14.96418.20 20.92
418.201.71
418.20 11.38418.20 23.20
418.202.04
418.20 60.04418.2038.32
0.0039.62418.20
NL: 3.05E9Base Peak F: FTMS + p NSI Full ms [300.00-1500.00] MS q1652
NL: 2.11E7m/z= 418.20-418.21 F: FTMS + p NSI Full ms [300.00-1500.00] MS q1652
q1652 #7896 RT: 20.85 AV: 1 NL: 2.49E7T: FTMS + p NSI Full ms [300.00-1500.00]
418.10 418.12 418.14 418.16 418.18 418.20 418.22 418.24 418.26 418.28 418.30 418.32 418.34m/z
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418.22
418.20
418.23 418.26
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How do you separate really complex mixtures?
25
Trace Analysis by Tandem Mass Spectrometry
26 758A . ANALYTICAL CHEMISTRY.V OL. 57. NO. 7, , l l ,NE 1985
Reac1on Ioniza1on
Collision-‐ Induced Dissocia1on (CID)
Tandem Mass Spectrometry
27
Collision-‐induced Dissocia)on (CID)
• Kine)c energy of parent ions is increased • Parent ions undergo energy conver)ng collisions • Parent ions fall apart into product ions and neutrals • Also referred to collision-‐ac)vated dissocia)on (CAD)
+ +
28
+
+
analyte ion m/z 400
matrix ion m/z 400
+
+
analyte product ion m/z 325
matrix product ion m/z 250
MS/MS (CID)
MS/MS (CID)
• Different compounds with same m/z but different structures • MS/MS allows interference-‐free quan)ta)on of analyte in matrix
Single Reac)on Monitoring (SRM)
29
30
Factors that determine what you can observe:
Where you look (samples) astronomy – region of space proteomics – sub frac1on of the proteome
The objects themselves (analytes) astronomy – luminosity, distance, composi1on proteomics – abundance, sequence, physical proper1es, PTM’s
Things that interfere (background) astronomy – sunlight, atmosphere, interstellar objects proteomics – abundant proteins, sample matrix, un-‐resolved proteins
Parallels between Astronomy and Proteomics
How you look (technology / method) astronomy – telescope, catalog space, search for phenomena proteomics – mass spectrometer, shotgun, targeted, differenBal mass spectrometry
31
Technology Enhances the “View” of the Cosmos
The Palomar telescope Courtesy California Ins)tute of Technology 200-‐inch telescope – 1949
M17 -‐ The Omega Nebula 7M X luminous than the Sun Credit & Copyright: 2MASS image mosaic by E. Kopan; courtesy the University of Massachuse8s and the Infrared Processing and Analysis Center/Caltech.
Charge Coupled Device "No method of advancing science is so produc)ve as the development of new and more powerful instruments and methods of research.” George Ellery Hale
Proteomic Technology -‐ Quadrupole Ion Traps
32
Electromagne)c Traps for Charged and Neutral Par)cles Wolfgang Paul, Nobel Prize, December 8, 1989
Proteomic Technology -‐ Modern Quadrupole Ion Traps Sequence Proteomic “Space”
33
Linear Quadrupole Ion Trap
MS/MS
Pep)de Sequence Informa)on
MS
Molecular Weight Informa)on
Proteomic Approach #1 -‐ Shotgun Proteomics
34
Modern Shotgun Proteomics Technologies
35
q1652 #6846 RT: 18.68 AV: 1 NL: 4.17E8T: FTMS + p NSI Full ms [300.00-1500.00]
300 400 500 600 700 800 900 1000m/z
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460.26324.87486.79
365.23
559.33 729.45417.24 651.34972.58
494.75
759.47 864.49 1025.50
q1652 #6847 RT: 18.69 AV: 1 NL: 6.51E6T: FTMS + p NSI d Full ms2 [email protected] [110.00-775.00]
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460.29347.20
633.33487.30443.26
374.21
597.29
q1652 #6848 RT: 18.69 AV: 1 NL: 1.07E6T: FTMS + p NSI d Full ms2 [email protected] [110.00-1160.00]
300 400 500 600 700 800 900 1000m/z
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357.07
665.23373.28682.25439.23 536.28 612.30
751.27
RT: 0.00 - 85.01
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38.6028.91
74.4629.09 38.72 61.21 63.5728.52 74.6764.5742.90
14.31 58.0047.8813.8955.76 75.1113.36
11.5377.38
NL:3.20E9Base Peak MS q1652
85 minutes and 39,556 spectra later…
Example Shotgun Proteomics Results 319 Proteins (2492) Pep)des Iden)fied from a Polyacrylamide Gel Band
36
Example Data Dependent LC-‐MS/MS Analysis
37
C:\Users\yatesn\Desktop\q1652 7/23/2012 11:04:10 AM
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38.60789.4328.89
687.86 31.81442.26
42.77910.45
63.56687.32
28.54687.86
74.431009.00
24.56844.44
61.19790.39
44.54644.86
64.59724.40
19.53609.31
14.32423.22 55.79
725.8611.53389.19
75.111086.88
7.35402.71
42.53910.4511.82
346.8419.73609.31
29.79371.73
36.35600.7724.02
632.36 74.37590.32
54.52120.08
63.46687.32
42.79663.42
11.69590.37
70.111044.56
38.06814.50
47.91635.31
1.30407.76 74.92
937.5178.70702.21
NL: 3.05E9Base Peak F: FTMS + p NSI Full ms [300.00-1500.00] MS q1652
NL: 1.22E10TIC MS q1652
q1652 #7735 RT: 20.54 AV: 1 NL: 2.66E6T: FTMS + p NSI d Full ms2 [email protected] [110.00-650.00]
150 200 250 300 350 400 450 500 550 600 650m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
288.20
403.23
502.30185.16
201.12 271.18 516.31158.09 386.20308.17120.08 229.12 446.26360.71 573.35485.27417.25
Examine the Data Collected From 20-‐21 Minutes
38
C:\Users\yatesn\Desktop\q1652 7/23/2012 11:04:10 AM
RT: 20.00 - 21.00 SM: 3B
20.0 20.1 20.2 20.3 20.4 20.5 20.6 20.7 20.8 20.9Time (min)
0
50
100
0
50
100
Rel
ativ
e A
bund
ance
20.00466.73 20.09
466.7320.17466.73 20.58
690.3320.23466.73
20.27466.73 20.97
544.7720.92397.70
20.21466.7320.16
482.2420.09143.12
20.08444.29
20.35120.08
20.32120.08
20.99684.38
20.59503.28
20.79563.31
20.54288.20
20.73120.08
20.68520.27
20.84218.15
20.47201.12
20.85186.12
20.38669.29
NL: 9.65E8Base Peak F: FTMS + p NSI Full ms [300.00-1500.00] MS q1652
NL: 8.42E9TIC MS q1652
q1652 #7734 RT: 20.53 AV: 1 NL: 2.08E8T: FTMS + p NSI Full ms [300.00-1500.00]
300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
461.24 672.38
512.78
690.33578.25
387.70
331.19 560.80921.46628.41
786.40720.41417.88
1024.55826.45 1120.60 1379.66885.48 932.52 1251.64
Tandem Mass Spectra of m/z 464.76
39
C:\Users\yatesn\Desktop\q1652 7/23/2012 11:04:10 AM
RT: 20.00 - 21.00 SM: 3B
20.0 20.1 20.2 20.3 20.4 20.5 20.6 20.7 20.8 20.9Time (min)
0
50
100
0
50
100
Rel
ativ
e A
bund
ance
20.00466.73 20.09
466.7320.17466.73 20.58
690.3320.23466.73
20.27466.73 20.97
544.7720.92397.70
20.21466.7320.16
482.2420.09143.12
20.08444.29
20.35120.08
20.32120.08
20.99684.38
20.59503.28
20.79563.31
20.54288.20
20.73120.08
20.68520.27
20.84218.15
20.47201.12
20.85186.12
20.38669.29
NL: 9.65E8Base Peak F: FTMS + p NSI Full ms [300.00-1500.00] MS q1652
NL: 8.42E9TIC MS q1652
q1652 #7737 RT: 20.54 AV: 1 NL: 7.51E5T: FTMS + p NSI d Full ms2 [email protected] [110.00-970.00]
150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
760.43
831.47
169.10 443.23 689.39618.35240.13141.10
465.76547.32319.11
434.23311.17175.12 573.35651.31476.24382.21 720.41285.13 920.47
219.15247.14
531.29352.04
604.29 798.38 890.42841.46
685.35
Data that Triggered the Acquisi)on of an MS/MS Spectrum
40
C:\Users\yatesn\Desktop\q1652 7/23/2012 11:04:10 AM
RT: 20.00 - 21.00 SM: 3B
20.0 20.1 20.2 20.3 20.4 20.5 20.6 20.7 20.8 20.9Time (min)
0
50
100
0
50
100
Rel
ativ
e A
bund
ance
20.00466.73 20.09
466.7320.17466.73 20.58
690.3320.23466.73
20.27466.73 20.97
544.7720.92397.70
20.56464.76
20.32464.72 20.89
464.7420.70464.78
NL: 9.65E8Base Peak F: FTMS + p NSI Full ms [300.00-1500.00] MS q1652
NL: 6.13E6m/z= 464.71-464.81 F: FTMS + p NSI Full ms [300.00-1500.00] MS q1652
q1652 #7734 RT: 20.53 AV: 1 NL: 2.08E8T: FTMS + p NSI Full ms [300.00-1500.00]
452 454 456 458 460 462 464 466 468 470 472 474 476m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
461.24
461.74
462.24460.77453.23 454.22 458.31 467.25462.74 475.24464.76 468.74455.22 474.30473.25465.75 470.74 476.22
Coun)ng Pep)des to “Quan)fy” Proteins?
41
Indx Accession Protein NumPep totalSpec UP003021 UP003022 UP003023 UP003024 UP0030251 A0AUZ9-1 Isoform 1 of Uncharacterized protein C2orf67 2 2 0 0 0 1 12 A0AV96-1 Isoform 1 of RNA-binding protein 47 1 1 0 0 0 1 03 A0AVG3 t-SNARE domain containing 1 1 2 1 1 0 0 04 A0JNW5-1 Isoform 1 of UHRF1-binding protein 1-like 1 1 0 0 0 1 05 A0N2G3 C-myc protein (Fragment) 1 1 0 0 1 0 06 A1A5D9-2 Isoform 2 of Bicaudal D-related protein 2 1 2 0 0 0 1 17 A1Z1Q3-1 Isoform 1 of MACRO domain-containing protein 2 1 1 0 0 0 0 18 A2A288-1 Isoform 1 of Probable ribonuclease ZC3H12D 1 1 1 0 0 0 09 A2CJ06 Dystrotelin 1 2 0 1 0 1 0
10 A2RTX5-1 Isoform 1 of Probable threonyl-tRNA synthetase 2, cytoplasmic1 3 0 0 1 1 111 A2VCQ4 PRKCSH protein (Fragment) 4 18 3 4 4 3 412 A2VEC9-1 Isoform 1 of SCO-spondin 1 2 0 1 0 0 113 A3KMH1-1 Isoform 1 of Uncharacterized protein KIAA0564 1 1 1 0 0 0 014 A4D1I1 Similar to ribosomal protein S3a; 40S ribosomal protein S3a; v-fos transformation effector protein 11 1 0 1 0 0 015 A4D1S5-1 Isoform 1 of Ras-related protein Rab-19 2 3 1 1 0 0 116 A4D1W8 Ependymin related protein 1 (Zebrafish), isoform CRA_b1 1 1 0 0 0 017 A4D256 Dual specificity protein phosphatase CDC14C 1 2 0 0 2 0 018 A4D997 Uncharacterized protein C9orf102 1 1 1 0 0 0 019 A4FU69-2 Isoform 2 of EF-hand calcium-binding domain-containing protein 51 1 0 1 0 0 020 A4UGR9-1 Isoform 1 of Xin actin-binding repeat-containing protein 21 1 1 0 0 0 021 A5D8V7 Coiled-coil domain-containing protein 151 1 1 0 1 0 0 022 A6NBZ8 Uncharacterized protein 1 1 1 0 0 0 023 A6NC98-1 Isoform 1 of Coiled-coil domain-containing protein 88B1 1 0 0 0 1 024 A6NDU8 UPF0600 protein C5orf51 1 1 0 0 1 0 025 A6NE01 Protein FAM186A 1 1 0 1 0 0 026 A6NEC2-1 Isoform 1 of Puromycin-sensitive aminopeptidase-like protein3 10 0 5 2 1 227 A6NEL2 Ankyrin repeat domain-containing protein 56 1 2 1 0 0 1 028 A6NH00 Olfactory receptor 2T8 1 1 0 0 1 0 0
MS separa)on of specific proteins?
42
43
QuanBficaBon: Pep&de concentra&ons are obtained by mul&plying the [IS] concentra&on by the signal ra&o of the analyte / internal standard
NH 2 COOH
Target Protein
QQQ DetecBon: Selected Reac)on Monitoring provides the high sensi)vity and amino acid selec)vity needed to detect pep)des in neat CSF
18 20 22 24 26 28 30 Time (min) 18 20 22 24 26 28 30 Time (min) 18 20 22 24 26 28 30 Time (min)
70 80 90 100
LC -‐ 18 20 22 24 26 28 30
Time (min) 0 10 20 30 40 50 60 70 80 90
100 0 10 20 30 40 50 60
Rela)ve Ab
undance
SRM
Analyte Signal = 200
Int. Std. Signal = 1000
Analyte Pep)de
AQTDIDSPQNLVTDR
NH 2 COOH
Standard Protein IS Pep)de
AQTDIDSPQNLVTDR*
Electrospray Ion
Source
CID Q1 Q3
521.7 757.6
Proteomic Approach #2 -‐ Selected Reac)on Monitoring Assays
Example Targeted Analysis Results
44
Proteomic Technology -‐ High Resolu)on Orbital Trap Mass Analyzers Expand Our “View” of the Proteome
45
Orbital Ion Trap More than 100,000 Observable Pep)de Species
Anne8e Michalski, Juergen Cox, Ma8hias Mann, Journal of Proteome Research, Feb 10, 2011.
C:\Xcalibur\data\114186 1/28/2004 7:58:10 PM PBI_167_01PBI_167_01
114186 # 2649-‐2723 R T : 24.01-‐24.50 A V: 37 NL : 1.02E 4F : ITMS + c NS I F ull ms [ 400.00-‐2000.00]
400 450 500 550 600 650 700 750 800 850 900 950m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
477.8879
456.1795
737.0342
736.9587
604.1706587.2521503.7772563.7734 607.8439521.9630448.4136 755.9727646.6342 722.0627 826.6938442.6017 666.4675 802.7613
910.4764895.1211839.1017 938.8213
114186 # 2649-‐2723 R T : 24.00-‐24.50 A V: 38 NL : 7.32E 4F : F TMS + p NS I F ull ms [ 300.00-‐2000.00]
400 450 500 550 600 650 700 750 800 850 900 950m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
737.0102
826.3724721.8766604.0323
563.5188477.5853 756.0177 802.9037607.8279
630.3329455.9778 895.4580549.3063 715.8750 910.9570521.7445646.2925448.2320 684.7227498.8625 980.9453827.8761 938.4314775.0247 866.4683
46
FTMS Technology – Observe More Pep)des
LTQ Low Resolution Detection
FTMS High Resolution Detection
C:\Xcalibur\data\114186 1/28/2004 7:58:10 PM PBI_167_01PBI_167_01
114186 # 2649-‐2723 R T : 24.01-‐24.50 A V: 37 NL : 3.37E 3F : ITMS + c NS I F ull ms [ 400.00-‐2000.00]
600 605 610 615 620 625 630 635 640 645 650m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
604.1706604.2482
607.8439
604.0094604.5195603.8953604.6180 646.6342627.7416607.9182 637.8765607.7653 620.4253 646.4821608.1470 628.1782 639.5774627.6029 637.7288618.3669 620.6226608.6675 630.3384623.3792 640.8938633.4860 648.3396603.7137 604.7827 641.3740626.5953613.8937 637.3624608.7394
615.3491 643.0638
114186 # 2649-‐2723 R T : 24.00-‐24.50 A V: 38 NL : 3.13E 4F : F TMS + p NS I F ull ms [ 300.00-‐2000.00]
600 605 610 615 620 625 630 635 640 645 650m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
604.0323
603.7816604.2830
607.8279
630.3329637.8310
608.3295604.5336 627.5958620.2660
646.2925638.3321630.8352627.3451620.7674 646.7942631.8751624.3262 640.9806604.7838 637.6454608.8308 618.2879 632.3765624.8277 641.3144 647.2949601.3377
47
FTMS Technology – Observe More Pep)des
LTQ Low Resolution Detection
FTMS High Resolution Detection
48
C:\Xcalibur\data\114186 1/28/2004 7:58:10 PM PBI_167_01PBI_167_01
114186 # 2649-‐2723 R T : 24.01-‐24.50 A V: 37 NL : 1.36E 3F : ITMS + c NS I F ull ms [ 400.00-‐2000.00]
617 618 619 620 621 622 623 624 625 626 627 628 629 630m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
627.7416
620.4253628.1782
627.6029618.3669
620.6226617.9780 623.3792618.4467
629.2044624.2963622.3300 628.2169
620.2675 623.3145618.5809 626.5953619.9922 626.0928625.2161 628.4862621.6592 627.4194623.2839
619.9135 629.2421624.3618621.1584619.6698
617.5070 629.8379
114186 # 2649-‐2723 R T : 24.00-‐24.50 A V: 38 NL : 1.10E 4F : F TMS + p NS I F ull ms [ 300.00-‐2000.00]
617 618 619 620 621 622 623 624 625 626 627 628 629 630m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
627.5958
620.2660
627.8463627.3451
620.7674
624.3262
623.3261618.2879 628.0974623.5773 628.3008623.0754 624.8277618.0365 618.5384 620.8176 626.2913
621.3194 625.9589622.3470 628.6346626.6250618.7888 625.3291 628.8247
624.3002
621.8208 622.8491
FTMS Technology – Observe More Pep)des
LTQ Low Resolution Detection
FTMS High Resolution Detection
49
C:\Xcalibur\data\114186 1/28/2004 7:58:10 PM PBI_167_01PBI_167_01
114186 # 2649-‐2723 R T : 24.01-‐24.50 A V: 37 NL : 1.17E 3F : ITMS + c NS I F ull ms [ 400.00-‐2000.00]
620.0 620.5 621.0 621.5 622.0 622.5 623.0 623.5 624.0 624.5 625.0m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
620.4253
620.6226623.3792
624.2963622.3300 623.3290623.8901623.6011620.2675 623.3145
621.6592 623.2839620.6414 622.9736622.6492620.9719 624.3618624.0942621.5428 623.2045622.7194 624.9265
621.7531 622.2361
621.8773
114186 # 2649-‐2723 R T : 24.00-‐24.50 A V: 38 NL : 1.00E 4F : F TMS + p NS I F ull ms [ 300.00-‐2000.00]
620.0 620.5 621.0 621.5 622.0 622.5 623.0 623.5 624.0 624.5 625.0m/z
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
620.2660
620.7674
624.3262
623.3261
623.5773623.0754 624.8277620.8176
621.2696621.3194 622.3470
623.8274623.7992
624.0492
624.3002
621.8208 622.8491621.6639
Mass Spectrometry – Detec)on and Separa)on of Ions
LTQ Low Resolution Detection
FTMS Detect attomole quantities of peptides Resolving power in excess of 200,000 Mass measurement error below 5 ppm
50
Sky and Telescope Ground-‐Based Image Archive Credit & Copyright: Image by Jay GaBany, San Jose, CA Observed with a 12-‐inch f/9 telescope and CCD camera 12 hour exposure from July 1st to 7th 2005.
“How” you look can define “What” you discover M51 – The Famous Whirlpool Galaxy
Supernova 2005cs
51
Differen)al Mass Spectrometry (dMS)
Key Benefits • supports clinically accessible samples • no pooling or chemical labeling • individual sample integrity retained • supports complex mul)-‐factor experiments
• no need for an)body reagents • quickly translates from animals to humans • )me matched with pharmaceu)cal research
An efficient MS based strategy for comparing complex biological systems. Un-‐biased analysis of all ions detected by full scan MS, not just ions that have corresponding MS/MS spectra and pep1de sequences.
Differen)al MS Workflow
52
Randomized Sample Set
Sample Processing prior to MS Analysis (e.g. Immunodeple)on or Ultrafiltra)on)
Desalt & run LC-‐MS
AD Control
Archived Human CSF (400 uL)
R.K*QLATK*AAR.-‐ Pep)de AA Sequence
dMS Feature
)me
m/z
CTRL
AD
Differen1al MS (dMS)
High Res MS
53
158360 # 2685-‐2809 RT: 24.81-‐25.34 AV: 3 NL: 5.29E5 F: FTMS + p NSI Full ms [ 300.00-‐2000.00]
300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 m/z
0 10 20 30 40 50 60 70 80 90
100
Rela)ve Ab
undance
544.298
913.487 722.371
787.882 969.482 1087.593 738.707
646.322 1094.057 867.410
552.295 457.245 959.447 521.786 670.337 639.631 592.338 1024.499 391.286
1107.560 452.238 481.238 881.943 695.631
828.440 776.410 919.446 342.265 982.878
The analysis of 1 CSF sample generates ~ 2000 high resolu)on spectra and that contain the m/z and intensity of more than > 100,000 pep)de ions.
Differen)al Mass Spectrometry (dMS) Reveals Features of Interest in High Resolu)on MS Data
dMS dMS
Summary
• Fundamentals • ESI • High and Low Resolu1on (Isotopes) • Tandem Mass Spectrometry • LC-‐MS and LC-‐MS/MS analysis • Data types
• Base peak chromatogram • Selected ion chromatogram • Mass spectra • Tandem mass spectra
• Proteomics • Shotgun proteomics • Targeted and global analysis of proteins • Trace analysis by selected reac1on monitoring • Differen1al mass spectrometry
Focus: Introduction to Mass Spectrometry and Methods for the Structural an Quantitative Analysis of Biomolecules.
54